Development of a functional vascular network is critically important for treatment of peripheral and cardiovascular disease and is a major problem limiting current tissue engineering strategies targeting repair and regeneration of damaged or diseased tissue. Recently endothelial colony forming cells (ECFCs), an endothelial progenitor cell population with high proliferative potential, have been shown to vascularize a type I collagen extracellular matrix (ECM) in vivo. In fact, ECFCs are the only cells that have been shown to possess direct in vivo vessel forming ability upon transplantation. This has generated much interest in the use of cord blood derived ECFCs for tissue engineering strategies. However, there is still a great need for refinement of a defined microenvironment to locally deliver ECFCs and guide vessel formation in vivo. The approach proposed here is the first to detail how the mechanical and microevironmental properties of a type I collagen ECM direct ECFC vascular network formation. By modulating matrix stiffness and fibril density, which have been shown to influence endothelial cell behavior in vitro, type I collagen 3D ECMs can potentially be engineered to support the formation of long lasting functional ECFC derived vessels. Importantly, this study will provide critical information for the development of vascularized tissue constructs that can be controllably delivered to ischemic areas and improve the efficacy of human umbilical cord blood derived ECFC therapies for human subjects. To accomplish these goals, we will pursue four aims. Aim 1) Determine how engineered 3D ECM design parameters (matrix stiffness and fibril density) and initial cell seeding density affect 3D vascular network formation by ECFCs in vitro. Aim 2) Investigate how specific engineered 3D ECM design parameters affect the ability of ECFCs to form a vascular network with functional anastomoses to host vessels when implanted in vivo. Aim 3) Characterize how engineered 3D ECM design parameters direct supporting cells to stabilize and prolong the function of ECFC derived vascular networks in vivo. Aim 4) Demonstrate that specific engineered 3D ECM design parameters affect the ability of ECFCs to accelerate wound healing in a full thickness skin wound murine model that more closely mimics a potential therapeutic application of locally delivered ECFCs embedded in a 3D ECM. The results obtained from the proposed studies will define the conditions that will optimize the in vivo blood vessel forming ability of cord blood-derived endothelial progenitors in a wound healing model that is relevant to human wound healing and provide a potential novel therapy to treat non-healing wounds. PUBLIC HEALTH RELEVANCE: